Confluence regulated adhesion molecules useful in modulating vascular permeability

ABSTRACT

The invention relates to new polypeptides in isolated form belonging to a subfamily of the human immunoglobulin superfamily, which polypeptide shows at least 70% sequence homology with the amino acid sequence of the murine Confluency Regulated Adhesion Molecules 1 or 2 (CRAM-1 or CRAM-2) as depicted in FIG.  3 , upper and second row, respectively, and antibodies thereto as well as their use in treatment of inflammation and tumors.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of Ser. No. 11/025,834, filed Dec. 30,2004, now U.S. Pat. No. 7,393,651, which is a continuation of U.S.application Ser. No. 09/524,531, filed Mar. 13, 2000, now abandoned, thedisclosures of which are hereby incorporated by reference in theirentireties, including all figures, tables and amino acid or nucleic acidsequences.

The present invention relates to the identification of a new subfamilyof vascular adhesion molecules and to the modulation of the function ofthese molecules for the treatment of various diseases.

Throughout embryonic and early postnatal development, endothelial cellsproliferate and differentiate to form new blood vessels viavasculogenesis and angiogenesis. In adult organisms the endotheliumdefines the blood-tissue barrier and consists of non-cycling quiescentcells. These polarized cells are linked to each other by tight junctionsand adherens junctions to form a continuous layer of cells. Thefunctions of the endothelial layer consist in the maintenance of tissuehomeostasis, fibrinolysis, coagulation, vasotonus, and leukocytetransmigration. All these properties are controlled by a fine tuning ofthe expression and the function of adhesion molecules.

Pathological situations such as inflammation, tumor growth, wounding orangiogenesis lead to a temporary change of the number and function ofadhesion molecules on the vascular endothelium and this results inaltered homeostasis of the vessel. As an example, tumors increase thelocal concentration of angiogenic factors which induces a switch fromnon-cycling quiescent endothelial cells to proliferating endothelium.The angiogenic switch is induced by several factors including IL-8,epidermal growth factor (EGF), vascular endothelial growth factor(VEGF), soluble VCAM-1, basic fibroblast growth factor (bFGF), and tumornecrosis factor (TNF). As a result, endothelial cells of existingvessels degrade the extracellular matrix (ECM) and invade thesurrounding tissue, which leads to vascularization of tumors.

During the angiogenic switch the pattern of endothelial gene expressionis modified. For example, treatment of endothelial cells with bPGF orTNFα results in a fourfold increase in α_(v)β_(j) integrin expression,an adhesion molecule implicated in endothelial cell migration. Inaddition, the angiogenic switch modifies the inflammatory response ofendothelium leading to an abnormal migration of leukocytes toward thetumors. Normally, leukocytes extravasate from the blood by adhering toand migrating through endothelium. These mechanisms occur in a multistepprocess that involves selectins, integrins and ImmunoglobulinSuperfamily adhesion molecules.

In tumor associated endothelium VCAM, ICAM, and selectins have beenshown to be downregulated. The down-regulation of these adhesionmolecules may represent a mechanism by which tumors avoid invasion bycytotoxic cells of the immune system.

It is the object of the present invention to search for new adhesionproteins of the Immunoglobulin Superfamily (Ig Sf), which aretranscriptionally regulated in endothelium under the influence oftumors.

It is a further object of the invention to define molecules derived fromthe new adhesion proteins for use in the treatment of variousindications, such as for example tumors and inflammation.

In the research that led to the present invention an experimental murinemodel was used for the identification of transcripts regulated duringthe co-culture of an endothelial cell line with melanoma cells. Torestrict the screening strategy to adhesion molecules of the Ig Sf, anew approach of RNA display termed “Targeted Differential Display” wasdeveloped. The novelty of the modified display technique resides in theuse of only one set of degenerated primers. As will be demonstrated inthe examples, it was surprisingly found that this leads to sufficientspecificity.

More specifically, partially degenerated primers (in the present casethe level of degeneracy is between 2048 and 4096 different forms ofprimers within one set), designed to target the conserved sequencesfound in the C2 domains of Ig Sf members were used to drive thePolymerase Chain Reaction (PCR) based Targeted Differential Displaytechnique (Samaridis & Colonna (1997) Eur. J. Immunol. 27, 660-665).

Based on this finding the invention provides a method for the specificidentification of differentially expressed DNA-sequences comprising theuse of Differential Display Reverse Transcription PCR, in which one setof partially or completely degenerated primers specific for the targetgene is used. One major limitation of the conventional RNA displaystrategy is the lack of specificity of the method. In the aim toincrease this specificity, the inventors in their search for otheradhesion molecules used degenerated primers targeting the sequencesencoding molecules with C₂ domains. This was achieved by the alignmentof C₂ domains of several Ig Sf adhesion molecules, and theidentification of a linear amino-acid consensus, surrounding thecysteine residue participating to the C₂ domain structure:Y-(RQYS)-C-x-A-S-N-x₂-G (SEQ ID NO:22). In a more general sense, thisapproach can also be used in the search for other sequences in which thereverse translation of one or more of the most frequent consensussequences is used to design the degenerated primers used fordifferential display.

This method allowed the identification of a transcript, downregulated inendothelial cells by confluency in the presence of melanoma or carcinomacells. The cDNA coded for a new molecule of the Ig Sf with the usualstructural features, and was named CRAM-1 for “Confluency RegulatedAdhesion Molecule”. The recent description of a structurally relatedmolecule, JAM, implicated in leukocyte transmigration, suggested theexistence of a new family of adhesion molecules in which JAM and CRAM-1were the prototypes. Sequence comparison with EST databases furthermoreallowed the cloning of CRAM-2, a third member of this molecular family.FIG. 1 shows the murine cDNA sequences encoding CRAM-1 SEQ ID NO:11 andCRAM-2 (SEQ ID NO: 12) proteins. In this application the names JAM andJAM-1, CRAM-1 and JAM-2 as well as CRAM-2 and JAM-3 are usedinterchangeably.

The comparative tissue distribution of the transcripts encoding JAM,CRAM-1 and CRAM-2 showed a preferential expression of these molecules inendothelial and epithelial compartments suggesting a role in themaintenance of cell-cell contacts. These cell-cell interactions ofquiescent endothelial cells regulate the vascular permeability, the cellcycle, and the leukocyte transmigration across endothelial wall.

To further elucidate the function and the interplay of the threemolecules, a molecular approach was used. To this end, chimericmolecules were constructed consisting of Flag-tag and Enhanced GreenFluorescent Protein (EGFP) sequences fused to a soluble or a membranebound form of CRAM-1, CRAM-2 or JAM (summarized in FIG. 2). Whentransfected into cell lines, the EGFP fusion products of CRAM-1 and JAMlocalized in cell-cell contacts, confirming a role of these molecules inthe cell-cell communication. In contrast, CRAM-2 was more widelydistributed on the cell surface. Moreover, the soluble construct ofCRAM-1 blocked transendothelial migration of leukocytes in vitro,whereas soluble JAM showed only marginal effect. Altogether, theseresults suggested a central role of this new subfamily of adhesionmolecules in the maintenance of vascular integrity and the function ofthe endothelial layer.

Based on these findings the present invention provides for new means ofcounteracting medical indications like chronic inflammation and tumordevelopment with reagents based on CRAM polypeptides.

More in particular, the present invention relates to a polypeptide inisolated form belonging to a subfamily of the human ImmunoglobulinSuperfamily, which polypeptide shows at least 70% sequence homology withthe amino acid sequence of the murine Confluency Regulated AdhesionMolecules 1 or 2 (CRAM-1 or CRAM-2) as depicted in FIG. 3 upper (SEQ IDNO:13) and second row (SEQ ID NO:14), respectively. FIGS. 4 and 5 showthe alignment on amino acid level between mouse (SEQ ID NO:13; top row)and human (SEQ ID NO:15) JAM-2 (CRAM-1) and mouse (SEQ ID NO:14) andhuman (SEQ ID NO:16) JAM-3 (CRAM-2), respectively.

The CRAM polypeptides found in the human or animal body are markers forgrowing cells. CRAM expression is upregulated in cells that are growing.

Disclosed herein are two new murine polypeptides that are member of thisfamily. Based on the sequence information of these polypeptides othermembers of the family can be identified by well known means such as PCR,crosshybridization on DNA libraries, crossreactivity of antibodies.

The sequence information can be either the amino acid sequence or thenucleotide sequence encoding the amino acid sequence.

More in particular, the invention thus relates to a correspondingpolypeptide in humans, comprising essentially the amino acid sequence asdepicted in FIG. 6B (top sequence, SEQ ID NO:15; bottom sequence, SEQ IDNO:16) or an amino acid sequence that is at least 70% homologousthereto.

In addition to using the sequence information of the two CRAM proteinsdisclosed herein for identifying other members of the family in otherspecies, like humans, the two proteins and their corresponding familymembers can also be used for the preparation of derived molecules, suchas antibodies directed against the (poly)peptides of the invention, orrecombinant equivalents of the proteins, optionally in soluble form, orpeptides comprising at least part of the amino acid sequence of thepolypeptides. Suitable parts of the amino acid sequence are especiallythe extracellular domains: VC₂, and the membrane proximal cytoplasmicsequence: A-[Y,Q]-[R,S]-[R,K]-G-[C,Y]-F (SEQ ID NO:26).

In addition to antibodies and (poly)peptide type derivatives, theinvention also relates to poly- or oligonucleotides having a sequencethat encodes a complete polypeptide or part thereof, which polypeptidehas an amino acid sequence that is at least 70% homologous to the aminoacid sequence of the CRAM-1 or CRAM-2 proteins as disclosed herein. Morein particular, the invention relates to nucleotide sequences that are atleast 70%, preferably at least 80%, more preferably at least 90%, mostpreferably essentially 100% homologous to the human DNA CRAM-1 sequenceas depicted in FIG. 6A (SEQ ID NO:17).

Such poly- or oligonucleotides may for example be RNA or DNA and can beprimers, probes, antisense RNA etc.

All such molecules can be used for modulating the function of theoriginal polypeptides found in the human or animal body or fordiagnosis.

Angiogenesis in for example tumors can be inhibited with antibodies.They can be used as targeting molecules for cells bearing the CRAMpolypeptides. The antibodies can act on their own or can be coupled toother molecules, such as toxins, radioactive labels, fluorescent labels,enzymatic labels, photo-activatable labels, but also to liposomes,cells, etc.

The labeled antibodies are particularly suitable for the diagnostic useof the antibodies, i.e. they can be utilized to locate angiogenesis in agrowing tumor. In addition, antibodies coupled to toxins or radioactivemolecules can be used to specifically kill the tumor from within bytargeting to the (growing) vessels in the tumor.

It was found that CRAM-type molecules were not detected in the normalvasculature except for lymphatics and the high endothelial venules inlymphoid organs such as lymph nodes and Peyer's patches. The advantagethereof is that the targeting of for example anti-CRAM antibodies can behighly specific to for example tumor cells thus avoiding undesirableside-effects.

Moreover, the (poly)peptides may also bind the molecule on angiogenicvessels and by that stimulate or inhibit angiogenesis.

Soluble (poly)peptide having essentially the same amino acid sequence asthe CRAM polypeptides can be used in the treatment of inflammationreactions of the vascular endothelium. It was found according to theinvention that the transendothelial migration of leukocytes can beinhibited by sCRAM-1-IG2Do or monoclonal antibodies against CRAM-1. Thisand similar molecules can therefore be used to quench or stimulate animmunological reaction such as found in inflammation.

The specific expression of the molecule on vascular cells of HEVs invivo which are specialized in lymphocyte migration argues for astimulating effect of CRAM on lymphocyte migration or vascularpermeability. This effect can therefore be due to the modulation ofmolecules normally involved in the sealing of the vascular bed (CRAM-1,CRAM-2, JAM, PECAM, VE-Cadherin) This finding is the basis for otherapplications of the invention involving the regulation ofinterendothelial junctions by delivering recombinant CRAM molecules(poly)peptides of the invention, or monoclonal antibodies againstCRAM-1.

Anti-CRAM antibodies can also be used to block cell-cell interactions ingrowing cells. This leads to disorganization of intercellular contactswhich are normally required for the barrier function of blood vessels.This finding may be used to increase the permeability of growing vesselsto increase the delivery of drugs to sites, such as growing tumors,post-menstrual uterus, etc. The disorganization of intercellularcontacts may therefore be used to block the development of tumor cellsbearing the antigen, such as angiomas (tumors originating from vascularendothelium) or some rapidly growing carcinomas.

For diagnosis use can be made of labeled antibodies but also of labeledoligonucleotides that are complementary to the CRAM DNA or mRNA found inthe endothelial cells expressing the CRAM protein(s).

The present invention will be further illustrated in the followingexample in which reference is made to the accompanying drawings, whichshow:

FIG. 1: Murine cDNA sequence encoding the CRAM-1 (SEQ ID NO: 11) andCRAM-2 (SEQ ID NO: 12) proteins. muCRAM-1 was subcloned in pcDNA3 vectorand sequenced using Sp6 and T7 primers. muCRAM-2 was obtained as IMAGEclone from EST library (Ac: AA690843 and W80145) and was sequenced inthe pT7T3-DPac vector using T7 and T3 primers.

FIG. 2: Schematic representation of the molecular tools used in theexample. The structure and important residues of the new family aredepicted in the upper top panel. The stars represent the putativephosphorylation sites in the cytoplasmic part of the three molecules.The second canonical Cys residue of the C2 domain is missing in the JAMsequence. Different chimeric molecules are represented below with theposition and the surrounding residues of the fusion sites. Part of themolecules originating from JAM, CRAM-1 or CRAM-2 sequences are shown inwhite background.

FIG. 3: Alignment of CRAM-1 (top; SEQ ID NO:13) and CRAM-2 (bottom; SEQID NO:14) amino acid sequences. Gaps are indicated as dashed.

FIG. 4: Alignment between murine (top; SEQ ID NO:13) and human (bottom;SEQ ID NO:15) CRAM-1 (JAM-2).

FIG. 5: Alignment between murine (top; SEQ ID NO:14) and human (bottom;SEQ ID NO:16) CRAM-2 (JAM-3).

FIG. 6: Nucleic acid sequence of human CRAM-1 (FIG. 6A; SEQ ID NO:17),complete amino acid sequence of human CRAM-1 (FIG. 6B (top); SEQ IDNO:15), and partial amino acid sequence of human CRAM-2 (FIG. 6B(bottom); SEQ ID NO:16).

FIG. 7: Targeted differential display using degenerated primers. (A):Nucleotide sequences of PCR primers (tayagntgynnngcytcyaa (SEQ ID NO:1);taycrgtgynnngcytcyaa (SEQ ID NO:2); taytaytgynnngcytcyaa (SEQ ID NO:3);tayagntgynnngcyagyaa (SEQ ID NO:23); taycrgtgynnngcyagyaa (SEQ IDNO:24); and taytaytgynnngcyagyaa (SEQ ID NO:25) encoding the sequencespresent in C2 Ig domains (SEQ ID NOS:18-20) are shown. Two primersencode the same sequence due to the codons encoding Ser residue. Thelevel of degeneracy is 4096 different forms for the primers encodingYRCXAS (SEQ ID NO:18) and 2048 forms for the others ((YQCXAS (SEQ IDNO:19) and YYCXAS (SEQ ID NO:20)). (B): The display of radioactive PCRproducts obtained with the YYCXAS1 (SEQ ID NO:20) primers is shown. Thelanes correspond to the display of PCR product run on cDNA obtained fromthe t-end endothelial cell line (lane t-end), the B16 melanoma cell line(lane B16), or the co-culture between the two cell lines (central lane).The arrow indicates the PCR product of interest obtained fromdownregulated transcript CRAM-1 under co-culture condition.

FIG. 8: (A) Nucleotide (SEQ ID NO: 32) and deduced amino acid (SEQ IDNO:33) sequence of Confluency Regulated Adhesion Molecule 1 (CRAM-1)cDNA. The putative hydrophobic signal peptide (first) and transmembraneregion (second) are underlined. Predicted N-glycosylation sites(strikeout), cysteines likely to form disulfide bonds (brackets) andSer/Thr/Tyr residues of possible phosphorylation sites (bold) areindicated. (B) Structural model for murine CRAM-1 protein. Extracellularpart showing a VH and a C2 like Ig domain with two putative N-linkedglycosylation sites. The arrow points to the region targeted by thepartially degenerated primers (YYCXAS1) (SEQ ID NO:20) used in theTargeted Differential Display.

FIG. 9: JAM (SEQ ID NO:21), CRAM-1 (SEQ ID NO:13), and CRAM-2 (SEQ IDNO:14) murine protein sequence alignment. The identical residues areboxed in black and the homologous residues are shaded in gray. Theoverall identity is 36% between CRAM-2 and CRAM-1, 31% between JAM andCRAM-1 and 33% between JAM and CRAM-2; the respective homologies are52%, 52% and 49%. The gaps are shown by dashes in the sequences. Thecanonical conserved residues (Cys and Trp) of the V and C2 domains aremarked by an asterisk.

FIG. 10: Expression of transcripts encoding JAM, CRAM-1 and CRAM-2detected by RT-PCR in different lines (A) or detected by Northern blotin various tissues (B) (A): RT-PCR is achieved on cDNA originating fromendothelial cell line treated by TNF (lanes 2 and 11 correspond to TNFtreated t-end) or not treated (lanes 3, 4, 6, 7, 9, 12 correspond tob-end.5, e-end.2, t-end V⁺⁺L⁻, t-end V^(low)L⁺⁺, TME and t-end,respectively). Lanes 5 and 10 correspond to the tumor cell lines B16(melanoma) and KLN205 (carcinoma). Lane 8 corresponds to the nontransformed thymic epithelial cell line MTE4-14. Lane 1 is the positivecontrol for JAM, CRAM-1 and CRAM-2 amplifications on the plasmidscontaining the cloned cDNAs. (B): Autoradiograph of P³² probehybridization to mouse Northern blot. The probes used for eachhybridization are indicated left. The hybridization signals for JAM andCRAM-1 are detected at the size of 2 kb.

FIG. 11: JAM-2 and JAM-1 localization to established cell-cell contacts.A: Immunocytochemistry was performed on paraformaldehyde fixed TME cellswith anti-JAM-2 (a) or anti-JAM-1 (b) antibodies. Arrows indicate thespecific localization of the proteins to cell-cell contacts. Bar, 10 μm.B: JAM-2-EGFP (a) and JAM-1-EGFP (c) chimeric molecules werespecifically localized to cell contacts between transfected cells. Theenrichment in EGFP recombinant proteins was not observed betweentransfected and non-transfected cells (arrowhead). Bar, 20 μm. C:Immunoprecipitation of JAM-2 after surface biotinylation of TMEendothelial cells. Anti-PECAM (lane 1) and anti-JAM-1 (lane 2)antibodies were used as negative and positive controls respectively forthe immunoprecipitation with CRAM-XIXH36 antibody (lane 3). Molecularweights are indicated on the right. D: Immunoprecipitation of EGFPrecombinant proteins from CHO transfected cells. Anti-JAM-2 (lanes 2, 3,6), anti-JAM-1 (lanes 1, 4, 5) were used to immunoprecipitate thebiotinylated lysates from untransfected (lanes 1 and 2), JAM-1-EGFP(lanes 3 and 4), or JAM-2-EGFP (lanes 5 and 6) transfected CHO cells.Molecular weights are indicated on the right.

FIG. 12: Migration of splenocytes across monolayers of TNF-activatedendotheliomas, in the presence or absence of the chemokine SDF-1. Threeendotheliomas were used: wild-type t.end.1, or t.end.1 transfected withthe cDNA encoding for CRAM-1 or CRAM-2. Two monoclonal antibodies weretested for their ability to affect transmigration, F-26 or H-26, bothrat IgG1 monoclonal antibodies directed against murine CRAM-1.

FIG. 13: CRAM-1 regulation in function of confluency. Thesemi-qauntitative PCR is driven using a mix of primers specific for HPRTand the CRAM-1 cDNAs. The PCR reactions are run on a 1.2% agarose geland stained with ethydium bromide. Lanes 1, 2 and 3, correspond to 100,50 and 10% confluency respectively. A weaker signal for CRAM-1 in the100% confluency (lane 1) is observed. The culture condition of theendothelial cell lines (t-end.1 and TME) on their own or mixed with thetumor cell line KLN 205 is indicated.

FIG. 14: Northern blot analysis of JAM-2 (a), JAM-1 (b) or β-actin (c)transcripts in mouse tissues. Results on embryonic post-coitum (pc) andadult mRNA preparations are shown. The sizes of the hybridizationsignals are indicated on the right.

FIG. 15: Immunohistological analysis of JAM-2, JAM-1, ZO-1 and PECAMexpression. Serial sections of kidney (a-d) or sections from mesentericlymph node (e-l) were stained with anti-JAM-2 (a, e, i), anti-JAM-1 (b,f, j), anti-ZO-1 (c, g, k) or anti-PECAM (d, h, l) antibodies. Eachseries of pictures (a-d, e-h, and i-l) were acquired with identicalsettings for the CCD.

FIG. 16: JAM-2 expression on endothelial cells. A: Cytofluorimetricanalysis of JAM-2, JAM-1, and PECAM expression on endothelial cell lines(tEnd.1, eEnd.2 and TME) or squamous carcinoma cell line (KLN 205).Dashed profiles represent the negative controls obtained with anantibody directed against CD4. B: Cytofluorimetric analysis of JAM-2 onfreshly isolated endothelial cells. Indicated organs were dissociated byCollagenase/dispase digestion, stained with DiIAc-LDL, CD31 andanti-JAM-2 or anti-JAM-1 as indicated. Histogram profiles were obtainedby gating endothelial cell population positive for DiIAc-LDL (FL-2) andCD31 (FL-3). Negative controls were obtained by omitting the primarytabs against JAM-1 or JAM-2.

FIG. 17: (A): JAM-2-EGFP localization during cell-cell contactformation. Single fluorescence pictures were collected every 3 min for 1hour during the monolayer formation of CHO cells transfected withJAM-2-EGFP. Pictures obtained during the first 18 mini are shown. Attime 0, asterisks identify the three cells present on the field. At time6, 12 and 18 min, arrows highlight the relocalisation of JAM-2-EGPP tothe newly formed cell-cell contact. (B): JAM-2-EGFP localization afterwounding. Arrows indicate the wounded side and arrowheads highlight themembrane processes rich in JAM-2-EGFP. Elapsed time is indicated on thepictures. Bar, 10 μm.

FIG. 18: JAM-2 expression decreases paracellular permeability. (A):Paracelluler permeability was evaluated by FITC-Dextran diffusion acrossnon transfected CHO cell monolayers, CHO cells transfected with Tac(huIL2Rα) or with the indicated EGFP fusion protein (JAM-1 or JAM-2).Transfection of JAM-2-EGFP or JAM-1-BGFP in CHO cells led to asignificant decrease in paracellular permeability (57.8%⁺/−4.9 and70.8%⁺/−3.6 respectively, p<0.0001), whereas transfection of Tac did notsignificantly affect the paracellular permeability (100.4%⁺/−4.4,p=0.9872) . Results were normalized to non-transfected CHO cells.

FIG. 19: Targeting of JAM-2-EGFP (A) and JAM-1-EGFP (B) to preexistingtight junctions. Confluent MDCK cells, stably transfected withJAM-2-EGFP (A), or JAM-1-EGFP (B), were stained with anti-occludin andanti-rabbit-Texas/Red. Series of pictures every 0.9 μm from basal toapical levels are shown for EGFP fluorescence (a) or occludin staining(b). The basal level on the left was arbitrarily defined such as theserial pictures comprise the tight junctional level on focus at +3.6 and+4.5 μm (fourth and fifth pictures to the right).

FIG. 20: Effect of soluble recombinant molecules on leukocytetransendothelial migration. (A): Transmigration is expressed as arelative index and normalized on the values obtained on the non-treatedt-end cell line (dashed line Index 1). Results obtained in the presenceof 1 μg sJAM-Ig2do (open squares) or in the presence of 1 μgsCRAM-1-Ig2do (filled circles) are shown. Index is calculated as a meanof five independent transmigration experiments. (B): Phenotype oftransmigrated cells is expressed as cell numbers calculated from thepercentages obtained by Facs analysis following staining with antiCD3-FITC and anti B220-PE. The stars indicate the experimental pointswith a significant difference to the control.

In the examples the terms JAM and JAM-1, CRAM-1 and JAM-2, as well asCRAM-2 and JAM-3 may be used interchangeably.

EXAMPLE Materials and Methods

Cell Lines

The thymic (tEnd.1), and embryonic (eEnd.2) endothelioma cell lines(Williams et al., 1989, Cell 57:1053-1063) were provided by Dr. W. Risauand Dr B. Engelhardt (Max Planck Institute, Bad-Nauheim, Germany). TheSV40 transformed lymph node endothelial cell line TME was provided by DrA. Hamann (Harder et al., 1991, Exp Cell Res. 197:259-267). The squamouscell carcinoma KLN 205, the CHO, the MDCK, and the myeloma cell lineSp2/0, were obtained from the American Type Tissue Culture Collection(ATCC). All cells, except CHO, were grown in DMEM (Gibco BRL, Paisley,Scotland), supplemented with 10% FCS (PAA Laboratories, Linz, Austria),2 mM Glutamine, 100 U/ml Penicillin and 100 U/ml Streptomycin (all GibcoBRL). CHO cells were grown in Nut.Mix.F-12 (HAM) medium supplemented asabove. Adherent cells were detached by washing with PBS/0.15 mM EDTAfollowed by 5 min incubation in trypsin/EDTA at 37.degree. C.

Display, Cloning and Sequence Analysis

For co-culture experiments, 5×10⁵ t.End.1 cells were grown together with2.5×10⁴ B16 F10 melanoma cells for 64 hours in 10 cm tissue culturedishes. As control, 5×10⁵ t.End.1 and 2.5×10⁵ B16 F10 cells were grownseparately under the same conditions resulting in confluent monolayersafter 64 hours. Total RNA was directly extracted in petri dishes withTrizol reagent following manufacturer's instructions (Gibco BRL,Paisley, Scotland). The cDNA was prepared from 5 μg of total RNA,employing oligo-dT (16-mer) primer and Superscript Reverse Transcriptase(Gibco BRL, Paisley, Scotland). The quality and quantity of cDNA werechecked by running 27 cycles of PCR on 1 μl of cDNA diluted 1:5, usingprimers specific for the housekeeping HPRT cDNA. Then the differentialPCR was performed with the following degenerated primers:⁵′TAYAGNTGYNNNGCYTCYAA³′ (SEQ ID NO: 1), ⁵′TAYCRGTGYNNNGCYTCYAA³′ (SEQID NO:2), and ⁵′TAYTAYTGYNNNGCYTCYAA³′ (SEQ ID NO:3) encoding for themost frequent amino acid sequences encountered in C₂ domains: YRCXAS(SEQ ID NO:18), YQCXAS (SEQ ID NO:19), and YYCXAS (SEQ ID NO:20). ThePCR conditions consisted of using: 2 μl of diluted cDNA; 2.5 μl of10.times. Goldstar PCR buffer; 2 μl of MgCl.sub.2; 2 μl of degeneratedprimers 0.3 mM; 0.5 μl of dNTP 0.1 mM; 0.1 μl of αP³³ dATP 10 mCi/ml(Amersham Pharmacia Biotech, Dubendorf, Switzerland); 15.65 μl H₂O; 0.25μl Goldstar Taq polymerase (Eurogentech, Seraing, Belgium).

The parameters for the PCR were as follows: 45 sec at 94° C., 90 sec at50° C., and 45 sec at 72° C. repeated 40 times. Formamide/EDTA loadingbuffer was added and samples were denatured for 2 min at 94° C. The PCRproducts were then separated on a 6% polyacrylamide gel, andautoradiographed using Kodak OM-Mat. The band intensities were compared.

Differentially expressed bands were cut from the dried polyacrylamidegel and fragments were retrieved by boiling and ethanol precipitation aspreviously described (Liang and Pardee, 1992, Science. 257:967-970). ThePCR products were then reamplified using increased concentrations ofdNTPs (0.2 mM instead of 2 μM) without P³³-ATP. The products ofre-amplification were cloned into pGem-T Easy Vector (Promega Corp,Wallisellen, Switzerland) as described previously (Sambrook, Fritsch,and Maniatis; Molecular cloning; 2^(nd); Cold Spring Harbor LaboratoryPress; 1989).

Nucleic acid sequences of two independent clones were determined usingthe Thermo Sequence Fluorescent Labeled Primer Cycle Sequencing Kit(Amersham Pharmacia Biotech, Dubendorf, Switzerland) and the LJ-COR DNAAnalysis System (MWG-Biotech GmbH, Ebersberg, Germany).

Identification of JAM-3

Sequence analysis and comparison were performed via the applicationsavailable on the ExPASy Molecular Biology Server i.e. Blast, Prosite,Swiss-Prot. Three different ESTs homologous to CRAM-1 were identified(Accession No. AA726206, AA052463 and AA175925). None of them encodedfor a full length transcript and comprised the initiating ATG sequence.Therefore, the 5′ coding sequence was obtained using the 5′RACE-PCRSystem for Rapid Amplification of cDNA Ends, Version 2.0 according tomanufacturer's instructions (Gibco BRL, Paisley, Scotland).

The three primers used were designed based on the EST sequences asfollows: 5′-GAGGTACTTGCATGTGCT-3′ (SEQ ID NO:4) for synthesis of thefirst strand, 5′-CGACAGGTGTCAGATAACA-3′ (SEQ ID NO:5) and5′-CACCCTCCTCACTCGT-3′ (SEQ ID NO:6) for the two nested PCRs. The5′RACE-PCR product was cloned into pGem-T Vector. To obtain the fulllength coding sequence for CRAM-1, the cloned 5′RACE-PCR product and theEST (accession No. AA726206) were digested with HpaI and NotIrestriction enzymes and ligated into pGem-t vector. Cloning of fulllength CRAM-2 was based on the same strategy of sequence comparison and5′RACE technique. The full-length cDNA encoding CRAM-2 was finallyobtained from ESTs accession numbers: AA690843 and W80145. These twoclones differ by the length of the 3′ untranslated region.

Northern Blot

Total mRNA from cells or tissues was extracted using Trizol (Lifetechnologies AG, Basel, Switzerland) according to manufacturer'sinstructions. Poly-A⁺ mRNA was extracted from 250 μg total RNA with theOligotex mRNA Purification Kit (Qiagen, Zurich, Switzerland). EmbryonicPoly-A northern blot was purchased from CLONTECH (P. H. Stehelin and CieAG, Basel, Switzerland). The riboprobes were prepared from pcDNA3 vector(Invitrogen, Leek, Netherlands), and comprised the sequences encodingfor the immunoglobulin domains of JAM-1 and JAM-2, or the full-lengthcoding sequence for .beta.-actin. Hybridization was performed at 62° C.in buffer containing 50% formamide. The blots were then washed twice(0.5.times.SSC, 0.1% SDS, 67° C.), and autoradiographed on Kodak X-Omatat −80° C.

Confluence Experiment

The effect of endothelial cell confluency on JAM-2 mRNA levels wasinvestigated. 2×10⁵ TME endothelial cells were cultured in 6, 10, and 15cm diameter culture dishes to reach different levels of confluency after64 hours ranging from 10 to 100%. The number of cells after. 64 hours,checked by trypan blue exclusion and counting, was the same in allcases, and was not related to the surface area of the petri dish.

Semi-quantitative PCR reaction or northern blotting were used todetermine relative amount of transcript in the various conditions. Forthe detection of the JAM-2 transcript, the ⁵′-GACTCACAGACAAGTGAC-³′ (SEQID NO:7) and ⁵′-CACCCTCCTCACTCGT-³′ (SEQ ID NO:8) primer pair was used,giving a 750 bp amplification product. As internal control, thefollowing primers specific for Hprt cDNA were used to amplify a 350 bplong fragment: 5′-GTTGGATACAGGCCAGACTTTGTTG-3′(SEQ ID NO:9) and5′-GAGGGTAGGCTGGCCTATAGGCT-3′ (SEQ ID NO:10).

Construction of Expression Vectors

The sequence encoding EGFP was subcloned from pEGFP-1 vector (CLONTECH,P. H Stehelin and Cie AG, Basel, Switzerland) into pcDNA3 using HindIIIand NotI sites, therefore named pcDNA3/EGFP. The 3′ restriction sites,HpaI and ScaI, found in the sequence encoding respectively thecytoplasmic domain of JAM-2 and JAM-1, were used to fuse the twosequences at the N-terminus of the EGFP in pcDNA3 vector (Invitrogen,Leek, Netherlands). The inserts encoding JAM-2 or JAM-1 were excisedfrom pGemt or pRc/CMV using SacII/HpaI or HindIII/ScaI digestions,respectively.

The coding sequences were then cloned in pcDMA3/EGFP vector digestedwith AgeI, blunted by fill-in and further digested with HindIII or SacIIenzymes. This resulted in fusion sites at amino-acid positions DGV₂₉₁for JAM-2 and QPS₂₈₅ for JAM-1. The transfection of CHO cells wasperformed am previously described (Ballestrem et al., 1998, J Cell Sci.111:1649-2-1658).

Stable transfectants used for permeability assays were selected bygrowing transfected CHO cells for two weeks in medium containing 1 mg/mlof G418. Resistant colonies were isolated and checked for EGFPfluorescence intensity by flow cytometry (FACScalibur apparatus, BectonDickinson, Mountain View, Calif.) and fluorescence localization bymicroscopy (Axiovert, Zeiss, Oberkochen, Germany).

Time-lapse video microscopy was performed using an Axiovert fluorescencemicroscope and Openlab software for image acquisition.

The mammalian expression vector pcDNA 3 (Invitrogen, Leek, Holland) wasmodified by integrating the Flag-Tag (G. Wiedle, Dep. of Pathology, CMU,Geneva) coding sequences. Flag-Tag constructs containing codingsequences for the soluble forms of the JAM, CRAM-1 and CRAM-2 proteinswere prepared by PCR. In all cases, the forward primers were designed tofit ATG initiation region. The reverse primers were designed in thesequences encoding the hinge region for the one Ig soluble form or inthe sequence encoding the region between the C2 and transmembranedomains for two Ig domains soluble molecules. All reverse primers had 31extensions containing a XbaI restriction site for direct in-framecloning in the Flag-tag modified vector. Pfu DNA polymerase was employedin the PCR to avoid frequent mutations (Stratagene, La Jolla, Calif.,USA). The PCR fragments were then digested with XbaI and cloned into thepcDNA-3 Flag-Tag vector, digested by EcoRI, filled by Klenow andfollowed by an XbaI digest.

Reagents and Immunofluorescence Analysis

The following monoclonal antibodies were used: anti-PECAM (GC51, ratIgG_(2a); EA-3, rat IgG₁) and anti-JAM (H202.106.7.4, rat IgG₁)(Malergue et al., 1998, Mol Immunol. 35:1111-1119.; Piali et al., 1993,Eur J Immunol. 23:2464-2471).

The panel of CRAM antibodies against JAM-2 was generated in thelaboratory using standard techniques, and recombinant soluble moleculeas immunogen (Aurrand-Lions et al., 1996, Immunity. 5: 391-405). Theselected hybridomas were screened by ELISA for the production ofantibodies recognizing specifically the recombinant soluble JAM-2molecule. Positive clones were further tested on CHO cells transfectedwith JAM-2 cDNA (not shown).

All CRAM antibodies are of the IgG₁ or IgG_(2a) isotype exceptCRAM-25F24, which is of the IgG_(2b) subclass. Antibodies were purifiedon Protein G sepharose columns (Pharmacia Biotech Europe, Dhendorf,Switzerland) according to the manufacturer instructions. CRAM-19H36 mAbwas used for immunoprecipitation, whereas CRAM-18F26 was the reagentused for immunohistochemistry. Similar results were obtained with C-4H31and CRAM-17D33 mAbs.

Immunofluorescence analysis was performed using secondary reagentscoupled to PITC or Texas Red (Jackson Immunoresearch, Milan AG, LaRoche, Switzerland) for cytofluorimetry and immunohistochemistry,respectively.

For immunohistochemistry, samples were fixed 5 min with cooled (−20° C.)methanol. Samples were rehydrated in PBS, gelatine 0.2%, Tween 20 0.05%,incubated overnight with the primary antibodies before washing, andrevealed with the appropriate secondary reagent coupled to Texas Red.For the analysis of fresh endothelial cells, dissociation of freshlydissected tissues was performed using collogenase/dispase digestion,according to established procedures (Kramer et al., 1984, J Cell Biol.99:692-698) . The dissociated cells were stained for 2 hours at 37° C.with DiI-Acetylated LDL (Molecular Probe Europe BV, Leiden, Netherlands)before staining with anti CRAM-19 and goat anti rat-FITC probe. Afterthree washes, cells were stained with biotinylated anti-CD31(Pharmingen) and streptavidine Red 670 (Life technologies AG, Basel,Switzerland).

JAM-1 or JAM-2 expression was analyzed on cells positive for the twoendothelial cell markers: Acetylated-LDL (FL-2) and CD31 (FL-3).Negative controls were obtained by omitting primary antibody.

Immunoprecipitations

Immunoprecipitations were performed as previously described(Aurrand-Lions et al., 1997, Cellular Immunology. 176:173-179) using 10mM Tris-HCl buffer pH 7.4, 150 mM NaCl, 0.5% Triton X100, 0.5% NP40,protease inhibitor cocktail (Roche Diagnostics Ltd, Rotkreuz,Switzerland) for lysis. After immunoprecipitation, SDS/PAGE, andtransfer to nitrocellulose membrane, the biotinylated proteins wererevealed using streptavidin coupled to peroxidase (JacksonImmunoresearch) and ECL (Amersham Pharmacia Biotech, UK).

Permeability Assays

Permeability was measured using Transwell chambers (6.5 mm diameter, PCfilters, 0.4 μm pore size, Costar Corp). In brief, 1×10⁴ transfected ornon-transfected CHO cells were cultured to confluency on filterspreviously coated for 30 min with 0.2% gelatin. After 5 days, the mediumwas changed for prewarmed Nut/F12 medium without FCS (500 μl in thelower chamber and 200 μl in the upper chamber). FITC-dextran (MW 38.900,Sigma Chemical Co) was added in the upper chamber at 1 mg/ml finalconcentration.

After 1 hour, chambers were removed and fluorescence was read directlyin the lower chamber using Cytofluor II. The mean fluorescence intensityof five independent chambers was calculated and compared using Statviewsoftware and t-test unpaired comparisons. To normalize experiments, thevalue of mean fluorescence intensity obtained with wild type CHO cellswas taken as 100%.

Transfection and Purification of Soluble Molecules

Transient transfection of 293 T. Bose 23 or stable transfection of CHOcells with the soluble Ig1Do and Ig2Do Flagtag/pcDNA-3 constructs werecarried out using Lipofectamine Reagent according to manufacturer'sinstructions (Gibco BRL, Paisley, Scotland). Following transfectionsupernatants were collected every two days during a ten day period. M2Beads (Kodak, New Haven, USA) covalently linked to anti-Flag antibodywere washed twice with PBS containing a Protease Inhibitor Mix(Boehringer Mannheim, Germany). The beads were then incubated at 4° C.For 3 hours with supernatant from the transfected cells. After fivewashes with PBS containing protease inhibitors, a column was packed withthe beads, and recombinant molecules were eluted with 10 mM glycinebuffer pH 3.4 according to the manufacturer. The eluted fractionscontaining the recombinant proteins were then concentrated onCentricon-10 (Millipore) and dialyzed against PBS.

Final protein concentration was determined using the Micro BCA assay(Biorad). The purified products were then submitted to a polyacrylamideSDS gel electrophoresis followed by coomassie blue staining to analyzetheir purity.

Transmigration Assays

Leukocyte transmigration across endothelial cells was performed aspreviously described (Wheerasinghe et al., 1998, J. cell Biology, 142;595-607). Briefly, 1×10⁵ t-end cells were cultured for two days intranswell units (polycarbonate filters, 5 μm pore size, costar) in thepresence of 1 μg of Ig soluble recombinant molecules sJAM 2do or sCRAM-12do. After two days, 1×10⁶ leukocytes obtained from lymph nodes andPeyer's patches were added to the upper compartment, and the number oftransmigrated cells was monitored during the experiment every hour.After 4 hours, transmigrated cells obtained from five independent wellswere pooled and submitted to cytofluorimetric analysis for B- and T-cellmarkers B220 and CD3. Results were obtained using a Facscalibur machineand the Cell-Quest analysis program (Becton-Dickinson).

For a transmigration assay with splenocytes, 1×10⁵ endothelial cellswere seeded in transwell units (polycarbonate filters, 8 μm pore size,costar) allowing the cells to form a monolayer over 18 hours. Medium inthe upper compartment was removed and 1×10⁶ leukocytes in 100 μl,freshly prepared from spleen by Ficoll centrifugation, were added to theupper compartment. SDF-1 was added to the medium (final concentration:40 nM) in the lower compartment to establish a chemokine gradientbetween lower and upper compartments. For the experiment withantibodies, purified antibodies 18-F26 or 19-H36 were added at theconcentration of 10 μg/ml in the upper compartment with splenocytes.After 4 hours, transmigrated leukocytes (in the lower compartment) wereharvested and counted. Results were expressed as % of input.

Results

Targeted Differential Display

The regulation of genes in endothelial cells depends on theirenvironment. The present invention was directed to the identification ofgenes that undergo regulation upon the contact of endothelium with tumorcells. For this purpose, an in vitro assay was developed using theco-culture of melanoma tumor cells (B16) with an endothelioma cell line(t-end). Total RNA extracted from the mix was used as template toprepare cDNA submitted to a differential PCR screen. The cDNA obtainedfrom the endothelial or melanoma cells cultured on their own were usedas controls. The three different patterns were compared to identify thetranscripts regulated by the co-culture condition. To limit the analysisto the sequences encoding for cell surface molecules of Ig superfamily,partially degenerated primers were used that target the sequencesurrounding the C-terminal cysteine of C2 domains in Ig superfamilymolecules. The most reproducible pattern of PCR products was obtainedusing primers that encode the sequence YYCxAS1 (FIG. 7A; SEQ ID NO:20).This improved method of RNA display technique was named TDD for“Targeted Differential Display”.

In repeated experiments of TDD, sixteen differentially expressed geneswere identified. Following cloning, nucleotide and amino acid sequenceanalysis, three of the sixteen PCR products were possible candidatesencoding for unknown members of the Ig superfamily. One of the threecandidates (CRAM-1) was chosen for further investigation. When grownseparately, t-end.1 endothelial and B16 melanoma cells expressed highlevels of the CRAM-1 transcript. However, under co-culture conditionsthe level of CRAM-1 expression was down regulated (FIG. 7B). Translationof a 350 bp long fragment corresponding to CRAM-1 showed the amino acidsequence YYCxAS (SEQ ID NO:20) indicating the endings of an Ig C2 domainfollowed by an open reading frame (ORF) containing a hydrophobic stretchof 18 amino-acids that signed a transmembrane region.

CRAM-1, a Member of the Immunoglobulin Superfamily

Sequence comparison between the PCR product sequence and nucleotidedatabases revealed homologous and identical sequences in mouse ESTsdatabases. The presence of ESTs indicated that the PCR productcorresponded to a sequence expressed in vivo. Three ESTs were found tocontain a 300 bp long sequence at their 5′ end, which was identical to300 bp in the TDD product. The 3′ ends of each EST contained a poly-Atail. In total the ESTs were 1270 bp in length and corresponded to the3′ end of the CRAM transcript. Since the 5′ end of the transcript wasmissing in the EST cDNA clone, it was obtained by 5′RACE-PCR. Theresulting 1980 nucleotide long full length coding sequence of thepostulated CRAM-1 cDNA is shown in FIG. 8A. There was a strong consensussite (GACATGG) for translation initiation 16 bp downstream from the 5′end, followed by a single ORF predicting a protein of 310 amino acid.The 31 amino-acid region subsequent to the potential initiatingmethionine, was characteristic of a signal peptide. The cleavage sitewas predicted to be at Ala 31-Val 32.

The putative structure of the murine CRAM-1 protein is shown in FIG. 8Band consists of an extracellular region with a variable heavy chain anda constant type 2 like immunoglobulin domain (Pfam, The Sanger Centreand Blast) with two potential N-linked glycosylation sites (aa 104 and192). The hydrophobicity analysis (Tmpred, ISREC) predicted atransmembrane region between positions 242-260. The postulatedcytoplasmic domain consisted of 49 amino acids and contained a number ofhighly conserved Ser/Thr and Tyr phosphorylation sites (FIG. 8A,residues in italic). The search of known patterns with the Prositeprogram identified the motifs. SSK/SYK as protein kinase C, SKQD/TSEE(SEQ ID NOS:27-28) as CK2 and KQDGESY/KHDGVNY (SEQ ID NOS:29-30 asTyrosine kinase phosphorylation signatures.

JAM, CRAM-1 and CRAM-2 Define a New Subfamily

Several proteins showed high homology to CRAM-1. Two members of the IgSuperfamily: Human A33 antigen and part of the mouse neural celladhesion molecule, N-CAM were found to have 41% and 46% homology withCR-1 respectively. JAM, another member of the Ig Sf, had a similarstructure a. CRAM-1 with 34% amino acid sequence identity, and 54%homology. The significant identity between JAM and CRAM-1 was used tofind a third closely related sequence in EST databases, namely CRAM-2.The identity between the three molecules suggested the existence of anew subfamily of molecules in the Ig superfamily (FIG. 9). The homologyconcerned not only the overall structure of V and C2 domains (C54 toC118 and C147 to C235 in FIG. 9) but also sequences inside thecytoplasmic domains. Interestingly, the most divergent regions betweenthe three molecules were found at the beginning of the V domain(position 40 to 60) and in the proximal cytoplasmic part (position 280to 300). The functions of these two regions correspond to sequencesinvolved in ligand binding and signal transduction in other members ofthe Ig superfamily suggesting a role of JAM, CRAM-1 and CRAM-2 incell-cell communication.

Tissue Distribution of JAM, CRAM-1 and CRAM-2 mRNA

Expression of the three transcripts in cells of different origin wasdetected using RT-PCR. All endothelial, epithelial and most tumor celllines tested, were positive, although at varying degrees for thedifferent transcripts (FIG. 10A). The highest expression level forCRAM-1 was found in the SV40 transformed REV cell line TME (lane 9), andin the embryonic endothelial cell line e-end 2-(lane 4). The CRAM-2 andJAM transcripts showed a more restricted distribution, and were found inadult endothelial cell lines together with the CRAM-1 transcript (lanes3, 7, 9 and 12). Notably, JAM and CRAM-2 transcripts were stronglydownregulated by TNF treatment of endothelial cells whereas the level ofCRAM-1 transcript remained unchanged (lanes 2 and 11). Interestingly, anembryonic endothelial cell line (lane 4) or an adult endothelial cellline representing an angiogenic variant of t-end (lane 6, failed toexpress JAM or CRAM-2.

The tissue distribution of JAM-2 transcript was explored by northernblotting and compared to JAM-1 (FIG. 14). The JAM-2 transcript was 2 kblong, highly expressed in embryonic tissue, and in Peyer's patches,lymph nodes, kidney, and testis of adult animals. A putative splicevariant of 1.8 kb was detected in testis. Expression of JAM-2 transcriptwas low in lung, liver, spleen, and thymus. The relative abundance ofJAM-1 and JAM-2 were compared during embryogenesis: the mRNA encodingJAM-2 was detectable as early as day 7.5 post coitum, whereas JAM-1 mRNAwas not detected at all during embryogenesis.

These results suggest that CRAM-1 is widely expressed duringembryogenesis and shows a restricted expression to epithelial orendothelial compartments in adult tissues. This is in agreement with theidea that it plays a role in the establishment and the maintenance ofthe polarized organization of cells.

JAM-2, a 45 kD Protein, Depending on Homophilic Interaction for itsLocalization to Cell-Cell Contacts

Since the HEV derived cell line TME expressed the highest level ofJAM-2, this endothelial cell line was used to further study thesubcellular localization of JAM-2, and to compare to that of JAM-1. Thelocalization of the JAM-2 protein on the surface of the endothelialcells was restricted to cell-cell contacts (FIG. 11A, a). The stainingfor JAM-2 was weaker than that observed for JAM-1 and less prominent inthe membrane extensions between cells.

Then it was investigated whether JAM-2 present at cell-cell contactsinteracted homophilically with JAM-2 or whether it interactedheterophilically with another molecule on the neighboring cell. For thispurpose the JAM-2 protein was fused to green fluorescent protein(JAM-2-EGFP), and the construct transfected in CHO cells. When CHO cellstransfected with JAM-2-EGFP cDNA reached confluency, JAM-2 was onlyobserved in cell-cell contacts where both cells expressed the protein(FIG. 11B), whereas the contacts between expressing and non-expressingcells were devoid of JAM-2 (FIG. 11B, a, indicated by arrow heads). Thesame result was obtained when cells were transfected with the chimericmolecule JAM-1-EGFP (FIG. 11B, b). This indicated that either JAM-2, orJAM-1, needed homophilic interactions to be localized at cell-cellcontacts.

To characterize JAM-2 biochemically, immunoprecipitations of JAM-2 orEGFP chimeric proteins, expressed by TME cell line or by transfected CHOcells, respectively (FIGS. 11C and D) were performed. The anti-JAM-2antibody, CRAM-19H36, immunoprecipitated a single band of 45 kD from TMEcells lysate (FIG. 11C, lane 3).

The apparent molecular weight was identical under reducing ornon-reducing conditions (not shown) and corresponded to the predictedmolecular weight deduced from the amino-acid sequence of JAM-2, eachN-glycosylation site accounting for 5 kDa. Immunoprecipitation of JAM-1using H202-106 anti-JAM specific antibody resulted in a single band oflower molecular weight (^(˜)42 kD, lane 2) that excluded a possiblecross-reactivity between anti-JAM-2 and anti-JAM-1 antibodies.

Immunoprecipitations of recombinant JAM-1-EGFP or JAM-2-EGFP proteinsafter surface biotinylation resulted in single broad bands of 70 and 73kD respectively, indicating that the molecules were expressed on thysurface of CHO transfected cells (FIG. 11D, lanes 4 and 6). Thesemolecular weights were expected since EGFP has a molecular weight of 28kD.

Tightness and Leukocyte Migration

In order to understand how molecules influence the integrity of theendothelial cell monolayer and how they regulate the function of thevascular endothelium, leukocyte trans-endothelial migration assays wereperformed in the presence of recombinant soluble JAM or CRAM-1.Endothelial cells were cultured for two days in the presence ofsJAM-Ig2Do or sCRAM-1-Ig2Do. The monolayer integrity was not affectedduring this period, probably due to the molecular redundancy of themechanism of cell-cell contact formation. The transmigration assay wasperformed in the presence of 1 μg of recombinant soluble molecules. Asshown in FIG. 20A, the number of transmigrating cells was poorlyaffected by the presence of sJAM-Ig2Do (open squares) during the firstthree hours. After four hours, the number of transmigrated cellsincreased when compared to the control (dashed line). In contrast, thepresence of sCRAM-1-Ig2Do (closed circles) strongly reduced the numberof transmigrating cells.

Since the leukocyte populations were heterogeneous, it was evaluated ifsCRAM-1-Ig2Do acted on a specific leukocyte subpopulation or whethertransmigration was blocked without specificity, For this purpose, thetransmigrated cells were labeled for the lymphocyte markers CD3 and B220(FIG. 20B).

Remarkably, sCRAM-1-Ig2Do specifically blocked the transmigration ofnon-lymphoid leukocytes, i.e. myeloid lineage cells (central panel,dashed columns). In contrast, sJAM-Ig2Do poorly increased the number oftransmigrating T cells (left panel, white column) without any effect onother cell subpopulations.

Furthermore, when endothelioma cells transfected with CRAM-1 were usedfor transmigration assay, an increased transmigration was observed (FIG.12), whereas the transfection of CRAM-2 was without effect on thetransmigration. When SDF-1 was added to the assay, the leukocytetransendothelial migration reached 20%. This was partially blocked bymonoclonal antibodies against CRAM-1.

These results indicate that the engagement of CRAM-1 between endothelialcells may regulate the function of the endothelial layer. It could beexpected that the molecules of this family will become a barrier whenendothelial cells reach confluency. To this end, the regulation ofCRAM-1 transcripts was explored in endothelial cells under differentculture conditions.

CRAM is Downregulated by High Confluency

Since the transcript that encodes CRAM-1 is not regulated by TNF, but isdownregulated when the endothelium was co-cultured with tumor cells, aconfluency assay was used to further explore this regulation. Under lowconfluency, the cells were actively cycling and CRAM-1 interactions didnot occur whereas under high confluency the cells divided less andCRAM-1 was engaged. The level of CRAM-1 mRNA expression was determinedunder various cell densities by semi-quantitative RT-PCR. As shown inFIG. 13, the expression level of CRAM-1 transcripts decreased whenconfluency was reached (lanes 1, 2, 3 in FIG. 13 correspond to 100, 50,and 10% confluency respectively). This effect was hardly detectable withthe t-end cells but was more pronounced with the TME cell line whichhighly expressed CRAM-1. The downregulation of CRAM-1 in endothelia wasalso enhanced when the endothelial cells were co-cultured with KLN 205carcinoma cells which themselves do not express CRAM-1. This confirmedthe link between CRAM-1 expression and the cell cycle since tumor cellswere described to increase the growth rate of endothelial cells uponcontact. It is noteworthy that the results obtained with KLN 205carcinoma cells was identical to the one used in our original screeningstrategy with the B16 melanoma tumor. This indicates a general mechanismby which tumors affect endothelial behavior.

JAM is Highly Expressed During Embryogensis, and Restricted to HEVs andEndothelial Cells Subpopulations in Adult Tissues

To better define the tissue distribution of JAM-2, immunohistologicalanalysis was performed on kidney and mesenteric lymph node sections(FIG. 15), which expressed the highest levels of JAM-2 transcript. Inthe cortical region of the kidney, a specific staining of intertubularstructures was detected with anti-PECAM (GC51) or anti-JAM-2(CRAM-XVIIIF26) mAbs, whereas anti-ZO-1 or anti-JAM-1 stainedpredominantly the tubular epithelial cells (not shown).

The inventors therefore focused their attention on vascular structures,which dip down into the medulla and correspond to radial veins or vasarecta endothelial structures. For this purpose, serial sections wereperformed and the vascular structures identified with the anti-PECAMstaining (FIG. 15 d). On the equivalent region of neighboring sections,linear interendothelial stainings were detected with anti-JAM-2,anti-JAM-1 or anti-ZO-1 (FIGS. 15 a, b and c, respectively). On sectionsof s enteric lymph node, typical staining of high enothelial venules(HEVs) was obtained with anti-JAM-2 mAb (FIG. 15 e). The HEVs were alsofound to express JAM-1, ZO-1 or PECAM (FIGS. 15 f, g and h), with subtledifferences in the subcellular localization of the stainings (FIG. 15e-h, insets). In the cortical area of the mesenteric lymph nodes, atypical staining of the subcapsular sinuses was observed with allantibodies (FIG. 15 i-l), corresponding to the staining of afferentlymphatic vessels. Thus, the staining with the CRAM-18F26 anti-JAM-2 mAbis restricted to certain endothelial cells or to structures closelyassociated with vasculature.

In order to clarify whether endothelial cells staining accounted for thepictures shown in FIG. 15, cytofluorimetry analysis of JAM-2 expressionwas performed on various cell lines or freshly isolated endothelialcells from dissociated tissues. Endothelial cell lines (tEnd.1, eEnd.2and TME) expressed low levels of JAM-2 on the cell surface and variablelevels of JAM-1 (FIG. 16A).

Cytometric analysis of freshly isolated endothelial cells was performedby triple staining of cell suspensions obtained aftercollagenase/dispase organ dissociation. Endothelial cells wereidentified by gating cells stained with both PECAM/CD31 andAcetylated-LDL (Voyta et al., 1984, J. Cell Biol. 99:2034). Staining forJAM-2 or JAM-1 on this double positive cell population is shown in FIG.16B. In kidney and Peyer's patches, all the isolated cells positive forCD31 and Acetylated-LDL were also stained for JAM-2, meaning that, atleast in these organs, endothelial cells expressed JAM-2 in vivo. Whenthe staining was performed on cells obtained from lymph node, JAM-2expression was only found on a cell subpopulation, reflecting a possibleheterogeneity of endothelial cell phenotypes within this tissue.

Altogether, the results of cytometric and immunohistochemical analysisshow that JAM-2 is co-expressed with JAM-1 by endothelial cells ofkidney, Peyer's patches and lymph nodes.

Dynamic Localization of JAM-2 to Cell-Cell Contacts

To dissect the mechanism by which JAM-2 was specifically localized tocell-cell contacts; time lapse video microscopy was used. The CHO cells,stably transfected with the fluorescent chimeric molecule, weretrypsinized and plated into chamber slides for imaging. After cellspreading, surface expression of JAM-2-EGFP was not uniform, but wasrather clustered at cell-cell contacts (FIG. 17A, cells depicted byasterisks). During the formation of new cell-cell contacts,relocalization of JAM-2-EGFP to cell junctions was observed and anintense fluorescence signal was detected at the novel contact pointbetween the cells forming the new cell-cell contact (arrows). Thechimeric protein was enriched in the membrane protrusions betweencontacting cells, leading to the “zipper like” pictures seen after 12 or18 min. Interestingly, the localization of JAM-2 at the primarycell-cell contacts was not lost during the formation of the new membranecontact (see upper left corner cell contacts). This finding indicatedthat JAM-2-EGFP was specifically relocalized to the new cell contact,and that, upon engagement, its localization was stable.

To further address the requirements for JAM-2 localization, time lapsevideo microscopy was performed after wounding the cell monolayer (FIG.17E). Cells at the wounded edge maintained JAM-2 at their intact contactsites (arrowhead), but lost JAM-2 localization at the wounded side(arrows), indicating that JAM-2 engagement by a ligand on the opposingcell was necessary to maintain its membrane localization. Over a periodof 90 min following wounding, cells bordering the wound began to migrateinto the wounded area. Interestingly, these cells maintained contactswith neighboring cells via membrane protrusions that were brightlyfluorescent, i.e JAM-2 positive (arrowhead). These results supported thehypothesis that JAM-2 homophilic interactions may play a role in theestablishment or maintenance of cell-cell contacts.

JAM-2 Increases Monolayer Tightness and Participates to Tight JunctionalComplexes

Since a number of molecules participating in cell-cell connection, havebeen shown to regulate the paracellular permeability of cell monolayers,it was tested whether JAM-2 could also affect this function.Transfection of JAM-2-EGFP reduced the paracellular permeability to FITCdextran and improved sealing of CHO cell monolayers by 42.5%; whereastransfection of the unrelated molecule Tac (IL2R α), did notsignificantly reduce the paracellular permeability of CHO transfectedcells (FIG. 18). The transfection of JAM-1-EGFP also reduced theparacellular permeability of CHO transfected cells monolayer.

These results raised the question whether the chimeric molecules wereable to participate to a subcellular specialized compartment such astight junctions in polarized epithelial cells.

To answer this question, the EGFP chimeric proteins were transfected inMDCK cells, and their subcellular localization compared with that of thetight junctional marker: occludin. As shown in FIG. 19A, when serialpictures every 0.9 μm were analyzed for EGPS fluorescence and comparedto occludin staining, JAM-2-EGFP was specifically enriched in cell-cellcontacts at the level of tight junction. At the basal level (left),intracellular dots of EGFP fluorescence were observed. A similaranalysis (FIG. 19B) of MDCK cells transfected with JAM-1-EGFP showed asimilar co-localization with occludin. Nevertheless, the distribution ofJAM-1-EGFP fluorescence was less continuous than that observed forJAM-2-EGFP at the level of tight junctions.

Discussion

This example reports the use of a new screening strategy to identifyregulated transcripts encoding members of the Ig superfamily of adhesionmolecules. Described here is the cloning with this method of the newmolecule CRAM-1 as a regulated transcript. The regulation observed inendothelial cells grown in the presence of tumors is confirmed bysemi-quantitative RT-PCR and is shown to be dependent on the growthphase of the cells. Due to differential expression under changing cellconfluency conditions, the name CRAM-1 for “Confluency RegulatedAdhesion Molecule-1” was adopted.

Also described herein is a closely related sequence to CRAM-1 namedCRAM-2. CRAM-1 and -2 represent the prototypes of a new subfamily ofadhesion molecules which also includes the recently described moleculeJAM (Chretien et al., 1998, Eur. J. Immunol. 28, 4094-4104; Malergue etal., 1998, Mol. Immunol. 35, 1111-1119).

CRAM-1 and JAM are preferentially expressed by endothelial andepithelial tissue at the cell-cell contacts and confer specialproperties to polarized layers. The effect of recombinant solublemolecules in a transendothelial migration assay and the regulation ofJAM, CRAM-1 and CRAM-2 show that these three molecules play an importantrole in the maintenance of vascular physiology.

The new screening strategy, named Targeted Differential Display (TDD),has proved to be an efficient technique in selectively amplifying cDNAof interest. TDD successfully exploited the use of partially degeneratedprimers to confer selective targeting to the conserved region,Y(Y/Q/R)CXAS (SEQ ID NO:31), of C2 like Ig domains. Repeated experimentslead to reproducible display patterns. Out of 16 differentiallyexpressed transcripts, three correspond to genes with significanthomology to conserved Ig sequences. This increase in specificity managesto overcome the major difficulties in the already known techniques ofclassical RNA fingerprinting and differential display. RNAfingerprinting has long been used for the identification ofdifferentially expressed genes. However, due to the sequence specificprimers employed, this method detects only the transcripts of selectedand already known proteins. On the other hand, RNA display employsrandom primers and involves the non-specific amplification oftranscripts. The aim in this case is to pinpoint any differences in mRNAlevels between two biological systems, which are submitted tocomparison. TDD is an advanced screening method that combines thespecificity of RNA fingerprinting with the degeneracy of DifferentialRNA Display resulting in selectivity. Due to the targeting of relatedtranscripts, this technique significantly reduced the time needed forscreening. The identification of new members of specific proteinfamilies, therefore, becomes possible. This is a substantial improvementof the reported non-specific screening strategies.

These common features were used to construct recombinant proteins inorder to study the functions of JAM, CRAM-1 and CRAM-2. In the presentexample, effects of sJAM-Ig2Do and sCRAM-1-Ig2Do are described in an invitro transmigration assay. Specific blocking effects on the migrationof myeloid cells could be observed with sCRAM-1-Ig2Do whereas sJAM-Ig2Doshowed only a small effect on lymphocytes.

JAM and CRAM-2 transcripts showed a similar tissue distribution andregulation of expression under the influence of TNF, indicating thatthey act by similar physiological mechanisms. In contrast, CRAM-1transcripts are not regulated by TNF but rather by the rate ofproliferation or the density of endothelial cells. In fact,overexpression of CRAM-1 transcripts in cycling cells and itsdownregulation in quiescent cells indicate that this moleculeparticipates in the establishment of a continuous monolayer. Itsfunction in confluent monolayers of cells is the maintenance of theendothelial cell layer and the related properties. Since differentleukocyte populations have to migrate to the site of immune response, itis thought that non-lymphoid cells migrate via a CRAM-1 dependantmechanism, whereas lymphoid cells migrate via JAM or CRAM-2. In thiscase the immune response can be modulated by using combinations ofdifferent soluble recombinant molecules.

1. An isolated nucleic acid molecule comprising: a) a polynucleotidesequence encoding SEQ ID NO: 13; b) a polynucleotide sequence thathybridizes under highly stringent conditions to a nucleic acid that isthe full complement of the nucleic acid encoding the amino acid sequenceas set forth in SEQ ID NO: 13, said highly stringent conditions includehybridization at 62° C. in buffer containing 50% formamide and a finalwash at 67° C. in 0.5×SSC and 0.1% SDS and wherein said amino acidsequence increases transendothelial migration of leukocytes; c) apolynucleotide sequence encoding a polypeptide comprising amino acids1-291 of SEQ ID NO: 13; d) a polynucleotide sequence encoding apolypeptide comprising amino acids 1-159 of SEQ ID NO: 13 and whereinsaid polypeptide inhibits transendothelial migration of leukocytes; e) apolynucleotide sequence encoding a polypeptide comprising amino acids25-241 of SEQ ID NO: 13 and wherein said polypeptide inhibitstransendothelial migration of leukocytes; f) a polynucleotide sequenceencoding a polypeptide comprising amino acids 1-238 of SEQ ID NO: 13 andwherein said polypeptide inhibits transendothelial migration ofleukocytes; g) a polynucleotide sequence that hybridizes under highlystringent conditions to a nucleic acid that is the full complement ofthe nucleic acid encoding amino acids 1-159 of SEQ ID NO: 13, aminoacids 25-241 of SEQ ID NO: 13, or amino acids 1-238 of SEQ ID NO: 13,said highly stringent conditions include hybridization at 62° C. inbuffer containing 50% formamide and a final wash at 67° C. in 0.5×SSCand 0.1% SDS and wherein said amino acid sequence inhibitstransendothelial migration of leukocytes; or h) a polynucleotidesequence encoding a fusion protein comprising: i) amino acids 25-241 ofSEQ ID NO:13; ii) amino acids 1-159 of SEQ ID NO: 13; or iii) aminoacids 1-238 of SEQ ID NO: 13; wherein said fusion protein inhibitstransendothelial migration of leukocytes.
 2. A transformed host cellcomprising a nucleic acid molecule according to claim
 1. 3. The isolatednucleic acid molecule according to claim 1, wherein said nucleic acidmolecule comprises a polynucleotide sequence encoding SEQ ID NO:
 13. 4.The isolated nucleic acid molecule according to claim 1, wherein saidnucleic acid molecule comprises a polynucleotide sequence thathybridizes under highly stringent conditions to a nucleic acid that isthe full complement of the nucleic acid encoding the amino acid sequenceas set forth in SEQ ID NO: 13, said highly stringent conditions includehybridization at 62° C. in buffer containing 50% formamide and a finalwash at 67° C. in 0.5×SSC and 0.1% SDS and wherein said amino acidsequence increases transendothelial migration of leukocytes.
 5. Theisolated nucleic acid molecule according to claim 1, wherein saidnucleic acid molecule comprises a polynucleotide sequence encoding apolypeptide comprising amino acids 1-291 of SEQ ID NO:
 13. 6. Theisolated nucleic acid molecule according to claim 1, wherein saidnucleic acid molecule comprises a polynucleotide sequence encoding apolypeptide comprising amino acids 1-159 of SEQ ID NO: 13 and whereinsaid polypeptide inhibits transendothelial migration of leukocytes. 7.The isolated nucleic acid molecule according to claim 1, wherein saidnucleic acid molecule comprises a polynucleotide sequence encoding apolypeptide comprising amino acids 25-241 of SEQ ID NO: 13 and whereinsaid polypeptide inhibits transendothelial migration of leukocytes. 8.The isolated nucleic acid molecule according to claim 1, wherein saidnucleic acid molecule comprises a polynucleotide sequence encoding apolypeptide comprising amino acids 1-238 of SEQ ID NO: 13 and whereinsaid polypeptide inhibits transendothelial migration of leukocytes. 9.The isolated nucleic acid molecule according to claim 1, wherein saidnucleic acid molecule comprises a polynucleotide sequence thathybridizes under highly stringent conditions to a nucleic acid that isthe full complement of the nucleic acid encoding amino acids 1-159 ofSEQ ID NO: 13, amino acids 25-241 of SEQ ID NO: 13, or amino acids 1-238of SEQ ID NO: 13, said highly stringent conditions include hybridizationat 62° C. in buffer containing 50% formamide and a final wash at 67° C.in 0.5×SSC and 0.1% SDS and wherein said amino acid sequence inhibitstransendothelial migration of leukocytes.
 10. The isolated nucleic acidmolecule according to claim 1, wherein said nucleic acid moleculecomprises a polynucleotide sequence encoding a fusion protein comprisingamino acids 25-241 of SEQ ID NO: 13, wherein said fusion proteininhibits transendothelial migration of leukocytes.
 11. The isolatednucleic acid molecule according to claim 1, wherein said nucleic acidmolecule comprises a polynucleotide sequence encoding a fusion proteincomprising amino acids 1-159 of SEQ ID NO: 13, wherein said fusionprotein inhibits transendothelial migration of leukocytes.
 12. Theisolated nucleic acid molecule according to claim 1, wherein saidnucleic acid molecule comprises a polynucleotide sequence encoding afusion protein comprising amino acids 1-238 of SEQ ID NO: 13, whereinsaid fusion protein inhibits transendothelial migration of leukocytes.13. The host cell according to claim 2, wherein said host cell comprisesa polynucleotide sequence encoding SEQ ID NO:
 13. 14. The host cellaccording to claim 2, wherein said host cell comprises a polynucleotidesequence that hybridizes under highly stringent conditions to a nucleicacid that is the full complement of the nucleic acid encoding the aminoacid sequence as set forth in SEQ ID NO: 13, said highly stringentconditions include hybridization at 62° C. in buffer containing 50%formamide and a final wash at 67° C. in 0.5×SSC and 0.1% SDS and whereinsaid amino acid sequence increases transendothelial migration ofleukocytes.
 15. The host cell according to claim 2, wherein said hostcell comprises a polynucleotide sequence encoding a polypeptidecomprising amino acids 1-291 of SEQ ID NO:
 13. 16. The host cellaccording to claim 2, wherein said host cell comprises a polynucleotidesequence encoding a polypeptide comprising amino acids 1-159 of SEQ IDNO: 13 and wherein said polypeptide inhibits transendothelial migrationof leukocytes.
 17. The host cell according to claim 2, wherein said hostcell comprises a polynucleotide sequence encoding a polypeptidecomprising amino acids 25-241 of SEQ ID NO: 13 and wherein saidpolypeptide inhibits transendothelial migration of leukocytes.
 18. Thehost cell according to claim 2, wherein said host cell comprises apolynucleotide sequence encoding a polypeptide comprising amino acids1-238 of SEQ ID NO: 13 and wherein said polypeptide inhibitstransendothelial migration of leukocytes.
 19. The host cell according toclaim 2, wherein said host cell comprises a polynucleotide sequence thathybridizes under highly stringent conditions to a nucleic acid that isthe full complement of the nucleic acid encoding amino acids 1-159 ofSEQ ID NO: 13, amino acids 25-241 of SEQ ID NO: 13, or amino acids 1-238of SEQ ID NO: 13, said highly stringent conditions include hybridizationat 62° C. in buffer containing 50% formamide and a final wash at 67° C.in 0.5×SSC and 0.1% SDS and wherein said amino acid sequence inhibitstransendothelial migration of leukocytes.
 20. The host cell according toclaim 2, wherein said host cell comprises a polynucleotide sequenceencoding a fusion protein comprising amino acids 25-241 of SEQ ID NO:13, wherein said fusion protein inhibits transendothelial migration ofleukocytes.
 21. The host cell according to claim 2, wherein said hostcell comprises a polynucleotide sequence encoding a fusion proteincomprising amino acids 1-159 of SEQ ID NO: 13, wherein said fusionprotein inhibits transendothelial migration of leukocytes.
 22. The hostcell according to claim 2, wherein said host cell comprises apolynucleotide sequence encoding a fusion protein comprising amino acids1-238 of SEQ ID NO: 13, wherein said fusion protein inhibitstransendothelial migration of leukocytes.